Viability and morphology of human fibroblasts in RGD-functionalized 3D alginate scaffolds Qualitative image analysis with focus on the effects of microstructuring

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NTNU Norwegian University of Science and Technology Faculty of Natural Sciences Department of Biotechnology and Food Science

Andrea Mandt-Utbøen Holtermann

Viability and Morphology of Human Fibroblasts in RGD-functionalized 3D- Alginate Scaffolds

Qualitative Image Analysis with Focus on the Effects of Microstructuring

Master’s thesis in Biotechnology Supervisor: Berit Løkensgard Strand Co-supervisor: Aman Sing Chahal September 2021

Master ’s thesis

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Andrea Mandt-Utbøen Holtermann

Viability and Morphology of Human Fibroblasts in RGD-functionalized 3D- Alginate Scaffolds

Qualitative Image Analysis with Focus on the Effects of Microstructuring

Master’s thesis in Biotechnology Supervisor: Berit Løkensgard Strand Co-supervisor: Aman Sing Chahal September 2021

Norwegian University of Science and Technology Faculty of Natural Sciences

Department of Biotechnology and Food Science

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Acknowledgements

This master thesis is a part of the Master's Degree Programme in Biotchnology at the Norwegian University of Science and Technology, and was carried out at the Department of Biotechnology and Food Science.

I want to give my deepest thanks to co-supervisor Aman Singh Chahal for patiently, and with dedication, teaching me everything I needed to know in the lab, and for supporting me all the way throughout the work with this thesis. I want to thank my supervisor Berit Løkensgard Strand for her unique ability to motivate and for always giving helpful advice and feedback. I also want to thank Daria Zaytseva-Zotova for introducing me to working with cells and for inspirational chats in the lab. Thanks to Astrid Bjørkøy for thorough training and help with the confocal microscope. It has been a fun, educational and challenging year. I also wish to thank study consultant Henrik Stamnes Dahl for providing clear information and being helpful with the practical and formal aspects with finishing this degree.

You have all been essential!

Lastly, I wish to thank my family and my friends for always being so caring and for putting up with me. I’m so grateful.

September 10^th, 2021

Andrea Mandt-Utbøen Holtermann

Per aspera ad astra

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Abstract

The use of three-dimensional (3D) cell culture rather than conventional two-dimensional (2D) cultures is of increasing interest, as it has shown to induce cell characteristics and functions of higher complexity that are more similar to those in vivo. This could offer more biologically relevant responses in cell studies, and therefore it is being actively adopted by fields such as cell biology, drug development, cancer research and tissue engineering. Many different 3D scaffolds exist, such as bulk gels, beads, macroporous sponges and foams, fibers and matrices.

Hydrogels, based on synthetic or natural polymers, is considered an attractive material due to their similarity to the extracellular matrix surrounding cells. Alginate hydrogels are one such material, popularly used due to its high biocompatibility and extensive ability to form gels of varying mechanical properties. Alginate gels are most typically formed via crosslinking with a divalent cation such as Ca²⁺. Unmodified alginate hydrogels typically do not support cell culture. To overcome this, chemical modifications can be applied to render this material as bioactive. However, mechanical properties such as stiffness, porosity and structuring are also of importance. For adherent cells such as fibroblasts, ligands for adhesion are crucial. These cells interact with their surroundings by binding to RGD-peptides, either in vivo to fibronectin of the extracellular matrix or in vitro to RGD grafted onto the culturing material. Attachment allows them to exert force on the material, and thereby also to exhibit movement and spreading by actin filament polymerization and myosin-actin contractility. Fibroblasts are common cells in research and are present in connective tissues throughout the whole body. In this study, the effects of alginate microstructures in 3D scaffolds on the viability and morphology of human fibroblastic cell types were investigated. These cells were primary normal dermal fibroblasts (NHDFs), cell line fetal lung cells (IMR-90) and cell line adult bone marrow cells (HS-5). The cells were cultivated for up to 21 days in a porous alginate foam and a homogeneous gel, both containing 1% (w/v) Ca²⁺alginate (75% functionalized with RGD-containing peptide GRGDSP), and analyzed after 1, 7 and 21 days using light microscopy, fluorescent labeling and confocal laser scanning microscopy and cytotoxicity tests based on LDH-release. No major differences in viability and cytotoxicity between foam and hydrogel were observed for any of the cell types. The NHDFs were generally more viable than the other cell types, and interacted more with the material, which could be related to them being primary cells. Morphological effects were seen in the form of actin extensions, with more organized cytoskeleton in cells exposed to the foam. Over time IMR-90 and HS-5 seemed to prefer cell-cell interaction, possibly due to inherent incompatibilities with the material properties.

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Sammendrag

Bruk av tredimensjonal (3D) cellekultivering istedenfor konvensjonell todimensjonal (2D) kultivering er av økende interesse, ettersom det har vist seg å kunne indusere mer komplekse egenskaper og funksjoner hos celler, som ligner mer på de i kroppen eller vev (in vivo). Dette kan bidra til responser av høyere biologisk relevans i cellestudier, og derfor er bruken av dette økende innen cellebiologi, legemiddelutvikling, kreftforskning og vevsteknikk. Mange ulike systemer for 3D-cellekultivering finnes, eksempelvis geler, mikrokapsler eller perler, makroporøse svamper og skum, fibre og andre 3D-matrikser. Hydrogeler, basert på enten syntetiske eller naturlige polymerer, er et attraktivt materiale på grunn av likheter med den ekstracellulære matriksen som finnes rundt celler i vev. Hydrogeler baser på alginat er populære på grunn av høy biokompatibilitet og god evne til å danne geler med varierende mekaniske egenskaper. Alginatgeler lages ofte ved tverrbinding med et divalent kation, som for eksempel Ca²⁺. Hydrogeler av umodifisert alginat er mindre anvendelige i cellekultur, men kjemisk modifikasjon kan gjøre materialet bioaktivt. Mekaniske egenskaper slik som stivhet, porøsitet og strukturering er også av betydning. For forankringsavhengige celler slik som fibroblaster er ligander som tillater forankring essensielt. Slike celler interagerer med omgivelsene ved å binde til RGD-peptider, enten til fibronektin i den ekstracellulære matriksen in vivo eller til cellekulturmaterialer modifisert med RGD. Binding tillater cellene å utøve kraft mot materialet, noe som tillater bevegelse og spredning av cellen ved polymerisering av aktin filamenter og kontraksjon utøvd av aktin og myosin i cellen. Fibroblaster finnes i kroppens bindevev, og er mye brukt i forskning. I denne studien undersøkes effektene av alginat-mikrostrukturer i 3D- kultivering på levedyktighet og morfologi hos menneskelige fibroblaster. Cellene som ble studert var primærceller fra dermis (NHDF), og cellelinjene IMR-90 og HS-5, henholdsvis med opphav i lungeceller fra embryo og stromale beinmargceller. Cellene ble dyrket opptil 21 dager i et porøst alginatskum og i en homogen gel, begge bestående av 1% Ca²⁺alginat (75%

funksjonalisert med RGD-inneholdende peptid GRGDSP), og analysert etter 1, 7 og 21 dager med bruk av lysmikroskopi, fluorescensfarging og konfokal laserskannemikroskopi, og test av cytotoksisitet baser på frigjort LDH. Ingen store forskjeller i levedyktighet og cytotoksisitet mellom skum og hydrogel ble observert i noen av celletypene. NHDF-cellene var generelt mer levedyktige enn de andre cellene, og interagerte mer med materialet, som muligens kan være relatert til at de er primærceller. Effekter på cellemorfologi ble observert i form av forlengelser av aktin, som i skum var preget av mer organisert cytoskjelett. Over tid foretrakk IMR-90 og HS-5 celle-celle-interaksjon, som kan skyldes inkompatibilitet med materialegenskapene.

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Abbreviations

2D Two-dimensional

3D Three-dimensional

AM Acetoxymethyl ester

AT Adenine-thymine

ATCC American Type Culture Collection

AU Adenine-uracil

bp Base pairs

CLSM Confocal laser scanning microscopy

-COOH Carboxyl

D1 Day 1 (after seeding cells and casting gels)

D21 Day 21

D7 Day 7

DAPI 4’,6-diamidino-2-phenylindole dihydrochloride DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid dsDNA Double stranded DNA ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid EthD-1 Ethidium homodimer-1

F Fraction

FBM Fibroblast basal medium FG Fraction of guluronate FGM Fibroblast growth medium FITC Fluorescein isothiocyanate

FN Fibronectin

FP Fluorescent protein

G α-L-guluronic acid

GAG Glycosaminoglycan

GC Guanine-cytosine

GDL Glucono delta lactone GFP Green fluorescent protein

GRGDSP Gly-Arg-Gly-Asp-Ser-Pro (RGD-containing peptide)

HA Hyaluronan

HBSS Hank’s Balanced Salt Solution

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HDF Human dermal fibroblast

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPMC Hydroxypropyl methylcellulose

HPV Human papilloma virus

HS-5 Cell line of human bone marrow stromal fibroblast-like cells HSC Hematopoietic stem cell

IC Inosine-uracil

IFF International Flavors & Fragrances

IMR-90 Cell line of normal human fetal lung fibroblast (Institute for Medical Research-90) INT Iodotetrazolium chloride

INT Iodonitrotetrazolium (tetrazolium salt) LAF Laminar air flow

LDH Lactate dehydrogenase

M β-D-mannuronic acid

MMP Matrix metalloproteinase MSC Mesenchymal stromal/stem cell Mw Molecular weight

NH Indole nitrogen

NHDF Normal human dermal fibroblast

-OH Hydroxyl

P Passage number

PBS Phosphate-buffered saline PEG Polyethylene glycol

PFA Paraformaldehyde

RGD Arg-Gly-Asp (tripeptide) RNA Ribonucleic acid

RT Room temperature

SMC Smooth muscle cell ssDNA Single stranded DNA

T75 Tissue culture flask (treated surface area 75cm²) TCP Tissue culture plastic

UP LVG Ultrapure low viscosity guluronate (alginate)

UV Ultraviolet

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1 INTRODUCTION

We live in a three-dimensional world. We can sense the ground under the soles of our feet, feel whether it’s raining, or the sun is shining on our heads. We can smell our food and recognize signs of danger. These are all important cues to tell us how to act and adapt in order to survive.

The cells we are built from live in such a world too – the three-dimensionality that is inherent to our tissues and organs. When cells are studied in vitro, they are removed from their natural microenvironment. While cells contribute to the construction of their environment, they also adapt themselves according to their surroundings. There is a mutual feedback interaction made possible through receptors on the cell surface, the cell’s way of sensing its world. Hence, when the cellular environment changes, the cell is bound to adapt to these changes. Traditionally, culturing of cells on two-dimensional (2D) tissue culture plastic (TCP) has been the standard.

However, current knowledge from research indicates culturing in three-dimensional (3D) materials might be more biologically relevant [1], especially for studying specific cell types or cell states [2]. Cells proliferate well in 2D, but there is a desire to allow the cells to express characteristics more similar to those in vivo. This can possibly be better achieved by making in vivo-like models by tailoring materials according to requirements of the cells. 3D materials that mimic the natural environment of cells offer a lot of possibilities within fields such as drug development and toxicology [3]. Due to this, there seems to be an ongoing shift from 2D cultivation on flat TCP to 3D encapsulation of cells in materials that more accurately mimic the natural cellular environment [3, 4].

1.1 3D cell culturing

To obtain a more biologically relevant model for cell studies, cells can be cultured in 3D instead of the traditional 2D monolayer on flat surfaces of glass or tissue culture plastic (TCP) [1, 3, 5- 7]. Several studies on the topic indicate that 3D cultured cells exhibit characteristics more similar to those in vivo (Figure 1) [2, 4, 8]. 2D cultivation is inexpensive and easy to use, but it puts cells in a state very different from their origin and exposes cells to stimuli unrelated to in vivo conditions [3, 9]. On flat surfaces, adhesive cells maximize their adhesion and have the tendency to look, behave and respond differently than in their natural cellular environment [5, 9]. Fibroblasts for example, which naturally are non-polar, will adopt a flat morphology with an artificial apical-basal polarity in 2D culture [6, 10], with most of the cell surface connecting either to the well plate or cultivation media [9]. 3D cultured cells seem to have more complex morphologies that are more comparable to their native appearance in tissue [9].

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Figure 1: Morphological differences between cells of bone (A, B and C), liver (D, E and F) and thymus (G, H, I), in their natural state, 2D cultivated and 3D cultivated. 2D cultivation gives the cells a stretched morphology, while 3D cultivation reveals a morphology closer to in vivo [8].

Cultivation of cells in 3D involves seeding of cells in a scaffold where they can interact with their microenvironment in all directions. The goal is to create a cellular environment that is more similar to that of the native tissue [5, 11], and by that providing tunable models that allow for studying interactions between cells and their environment [1, 3, 5]. 3D culturing can be a useful tool in e.g. cell biology [2, 12], cancer research [13] and tissue engineering [5].

Furthermore, drug development and toxicology studies can be improved by the potentially more relevant cellular responses [1, 3, 5]. 3D models can also lead to a reduced need for animal trials [5], which are not always able to predict the responses of human cells [14].

Several studies suggest that 2D is not sufficiently selective to dissect differences between cells [2, 12]. By cultivation in 3D collagen gels, differences between dermal fibroblasts from different patients that could not be observed in 2D culturing, have been observed [2]. Another study indicated that 3D culturing of normal and malignant breast cells revealed phenotypical differences that were not expressed when grown in 2D [15]. 3D cultivation has also shown to promote normal growth of epithelial cells with epithelial polarity [6], and exhibition of natural phenotypes in hepatocyte primary cells [3]. The higher relevance of information from 3D cultured cells compared to 2D has also been shown in gene expression profiles [7, 9]. This has been seen in breast cancer research [7, 16], and in studies on fibroblasts [17]. In a study by Li

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et al. (2003), gene expression profiles of smooth muscle cells (SMCs) grown on a 2D surface and in a 3D collagen matrix showed to differ in expression of 99 genes [17, 18].

1.1.1 The gel-like extracellular matrix

In their natural state, most cells are surrounded and structurally supported by the extracellular matrix (ECM) (Figure 2), which is a hydrated gel-like network of proteins and polysaccharides.

It comprises of glycosaminoglycans (GAGs), such as hyaluronan (HA), fibrous proteins, such as collagens, and glycoproteins such as fibronectin (FN). The ECM allows cells to attach and communicate with their environment and other cells [1, 3, 19]. Interaction between the ECM and cells is important for regulation of cell survival, proliferation, morphology, migration [4, 5, 19-21] and the generation of traction forces [22]. The components of the ECM are produced and secreted by the cells in the matrix. Important ECM-producers are epithelial cells and mesenchymal cells such as fibroblasts. Cells organize, remodel and degrade their ECM, mainly through the integrin attachment. The ECM composition is adapted to the functions of the cell types, and therefore varies between different tissues [1, 4, 19].

GAGs are often linked to proteins, forming proteoglycans that serve as a hydrated polysaccharide “gel” in which the other ECM-components are embedded in. This gel-like mesh permits diffusion of nutrients, hormones, cytokines, enzymes, and other metabolites [1, 19].

FNs have an important role in organizing the matrix and in the attachment of cells to the matrix [1, 19, 23], and they also function as tracks which the cells can use to migrate [19]. The FN protein contains a peptide sequence of Ar-Gly-Asp (RGD), which functions as a ligand and a binding site for integrins on the cell surface. Integrins are transmembrane protein dimers that function as cell receptors [19]. In addition to attaching the cell to the matrix, integrins transmit both mechanical and molecular signals both ways through the plasma membrane.

Intracellularly, the integrins are connected to the cytoskeleton, which mainly consists of actin filaments [1, 19, 24].

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Figure 2: Main composition of the natural extracellular matrix (ECM). A sequence of amino acids (RGD) in FN functions as a ligand for the transmembrane cell receptor integrin, allowing cell attachment and signaling to the cytoskeleton [1, 19, 23]. Illustration from Biological Science, 2005 Pearson Prentice Hall, Inc [25, 26].

1.1.2 Scaffolds for 3D culture

As 3D cultivation is a growing field, there exists a lot of different scaffolds, both based on synthetic, semi-synthetic and natural materials [4, 5]. There are 3D systems for cells both with and without a matrix scaffold [27]. In systems without a scaffold, the cells have to create their own ECM without physical support or porosity, such as in tumor spheroids [28]. 3D scaffolds however, may include different types of approaches to imitate the ECM [27]. Common formats of scaffolds include gels, beads, matrices, fibers, and porous materials [9, 29, 30]. Hydrogels have been applied widely to create 3D scaffolds for cells, due to their similarities with the gel- like ECM [5, 31, 32]. These are hydrated polymer networks [33] that exist as scaffolds in different sizes and shapes [29]. They can be made from polymers that are either synthetic, such as polyethylene glycol (PEG), or natural, such as hyaluronan (HA) or alginate [31]. Entrapment of cells in a 3D hydrogel often involves the cells being mixed in a precursor solution, then entrapped in the gel by either non-covalent or covalent cross-linking [3, 31]. In addition to being similar to the ECM, hydrogels are easy to handle and their properties such as degree of diffusion and degradation can be controlled through processing [34]. Regarding natural polymers for hydrogels, these can be derived from either animal tissue [31] or non-animal sources [27, 35].

Animal-tissue-derived hydrogels are naturally compatible with animal cells [31], seen for instance with laminin-rich ECM gels [36] and Matrigel^TM. The latter is a 3D gel that contains matrix proteins and glycoproteins from mouse sarcoma cells, and that showed to successfully

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support cultivation of cells exhibiting natural morphology and behavior [4, 30, 31, 37].

However, with animal-derived materials there are concerns regarding availability, batch-to- batch variation, the possibility of pathogen transmission and immunogenicity, which makes such materials less attractive [4, 31]. This draws attention towards non-animal derived natural polymers, such as polysaccharides like alginate, as a viable alternative. Alginate hydrogels have been widely applied in the field of 3D cell culture and tissue engineering due to their biocompatibility and favorable gelling properties [27, 35].

Mimicking the ECM is an important part of creating suitable 3D scaffolds for cells [5], and has led to extensive ongoing research on the subject [4]. The composition and stiffness of a mimicked ECM can be adapted to imitate the natural cellular environment of the cell type of interest. In that way, the cell’s native morphology and function can be better achieved [3] and understood [38]. This involves creating functional materials with suitable physical properties, e.g. regarding degree of stiffness and porosity, and with modifications that are able to support attachment and enable interaction [24].

A variety of cell types are often referred to as being adhesion dependent. This implies that anchoring of the cell to a solid or semi-solid interface is crucial for its survival [3, 9, 24]. Cells of connective tissue, including soft tissues and bone, are adherent cells that require attachment to the ECM in order to survive, spread and proliferate [3, 39]. In such cells, lack of attachment often results in cells gravitating towards apoptotic programming [24, 40]. Such apoptosis can depend on lack of attachment sites, the present integrin types and changes in the mechanical force of surroundings [41]. The ECM is a dynamic environment that undergoes phases of formation and degradation. Cells themselves form their cellular environment [1, 4, 19] by secreting ECM components as well as degrading enzymes such as matrix metalloproteinases (MMPs) and GAG-hydrolases. Therefore, the degradability of the hydrogels used for cell immobilization matters [3]. Hydrogels can be produced with varying degree of stability. This can be adjusted by modifying the gels with segments that are susceptible to hydrolytic or enzymatic degradation [33, 34]. In a study by Bott et al. (2019), human dermal fibroblasts (HDFs) cultivated in PEG-hydrogels modified with MMP-degradability and RGD-peptides for cell adhesion exhibited their tissue-typical spindle-shape morphology and created cell-cell networks. Cells in gels with only the RGD-grafting remained mostly round with only sporadic spreading, and cells in gels without RGD did neither spread nor proliferate. However, in spite of being functionalized for RGD-binding and MMP-cleavage, high material stiffness with elastic modulus more than 1200 pascals (Pa) seemed to impede the fibroblast’s ability to move

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and proliferate, acting as a barrier [2]. Also, regarding gel stiffness, HDFs seeded on top of a HA/FN-based gel have shown to adopt a more stretched and organized cytoskeleton and proliferate more in stiffer substrates (4270 Pa), similar to on TCP. However, cells seeded on softer materials (550 Pa and 95 Pa) migrated faster and had a less dense cytoskeleton with signs of buckling and spots [38]. A similar observation of coherence between material stiffness and cytoskeletal organization has been reported with hepatocytes [42].

For hydrogels, it is desired that they have pores that allow transport of small molecules, oxygen, nutrients, and waste, while preventing diffusion of large molecules. The diameter of pores of the surface of a hydrogel crosslinked with calcium usually varies between 5-200 nm [43].

Macroporous scaffolds such as sponges and foams give more surface area for attachment and proliferation [30, 31, 44], and have shown to be especially beneficial in growing bone cells [44]. Such scaffolds usually allow a well spread, spatially organized cell seeding. However, these scaffolds are often considered to be between 2D and 3D, as the increase in surface area gives similarities with flat 2D cultivation [31].

To find an appropriate 3D system for cell studies, one must aim for a culture system that allows for an accurate and accessible investigation of the cell in such materials, while also considering the cell requirements and study aim. Some of the methods that have been mentioned here are 2D culturing on TCP or on top of a gel, 3D culturing by entrapment inside a gel or gel beads, 2.5D culturing in macroporous scaffolds and spheroid 3D culture without scaffold.

Mesenchymal stromal cells (MSCs) have shown to have improved maintenance of functional characteristics in culture when hydrogel cell-matrix interactions are provided, compared to TCP culture [45, 46]. They have been tested in many different materials, and have shown improved secretion, migration, proliferation and cell-cell interaction in a macroporous scaffold with average pore size around 120 mm, compared to in a nanoporous hydrogel (average pore size 5 nm) [46, 47]. In a review by Weschler et al. (2021) on how hydrogel material properties affect the secretome of MSCs, an overview on different current hydrogels scaffolds and their advantages in MSC culturing is provided (Figure 3) [46]. The figure shows some important cons of culturing on TCP, on top of hydrogel, inside of hydrogel, in microspheres, such as beads, in porous scaffolds and spheroids [46].

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Figure 3: Different culture systems compared by their ability to improve certain desirable properties, graded by no improvement (-), some improvement (↑) and higher improvement (↑↑). The qualitative assessment is based on results from cultivation of MSCs. Figure by Wechsler et al. (2021) [46].

1.2 Alginates as 3D scaffolds

Alginates are natural biopolymers in the form of polysaccharides. They can be extracted industrially from brown seaweeds such as Laminaria spp., Ascophyllum nodosum, Ecklonia spp., Macrocystis spp. and Lessonia spp. [48], but are also produced by some bacteria like Pseudomonas sp. and Azotobacter vinelandii [7, 49]. Isolated alginates are converted into water-soluble alginate salts, such as sodium (Na) alginate [48]. Due to their gelling abilities, alginates are widely used in pharmaceuticals and in foods [7] as thickeners and gelling agents [48]. Alginates have shown to be biocompatible and are therefore suitable as biomaterials in biomedical applications. Alginates for such purposes are ultra-purified, removing any contaminants and leads to materials of low toxicity and immunogenicity [7, 50]. Some applications that alginate-based biomaterials have been used for are immobilization of cell and enzyme systems [51], proliferation of mammalian cells [7], tissue engineering [52], cell encapsulation [53, 54] and drug or cell delivery [55].

Alginates are linear, unbranched polymers of the uronic acids β-D-mannuronic acid (M) and α- L-guluronic acid (G), linked by (1,4)-glycosidic bonds. G is the C5-epimer of M [56]. The polymers occur as long chains with a pattern of blocks of M, blocks of G and sequences with alternating M and G (Figure 4) [48, 57-60]. The order and fraction (F) of M, G or MG depends on the source the alginate was isolated from [56].

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Figure 4: Chemical structure of a sodium alginate chain built from monomers of α-L-guluronic acid (G) and β-D-mannuronic acid (M). Figure retrieved and modified from Alginates: Biology and Applications (2009) by Rehm [49].

Soft alginate-based hydrogels can be prepared by cross-linking the alginate chains with divalent cations such as calcium (Ca²⁺) barium (Ba²⁺) and strontium (Sr²⁺) [7, 48, 61]. Most divalent cations can be used, except for magnesium (Mg²⁺) [60]. Alginate in combination with Ca²⁺for preparation of viscous solutions and gels is widely used [56]. When Ca²⁺ ions react with alginate, they attach to G-blocks in different alginate chains and forms interactions between them, creating a network [48, 56]. The calcium ion exchanges the sodium ion, because its binding to the chain is of higher affinity [48]. However, also blocks of MG are able to form stable crosslinks with Ca²⁺, both with other blocks of MG and of GG, as shown in Donati et al.

(2005) [62]. Grant et al. (1973) visualized the interaction between polysaccharides and divalent cations, such as alginate and Ca²⁺ ions, in the “egg box model” (Figure 5), the ions being eggs placed in the empty spaces of the alginate chain egg-box [48, 63].

Figure 5: The egg-box model. Divalent cations (dots) fit in the alginate chains as eggs in a box. Original drawing from Grant et al. (1973) [63].

To make a gel, the water-soluble alginate salt must first be hydrated [48]. Then, there are different methods for gelation. Two main methods based on ion crosslinking are internal setting and external setting. In the latter, there is an external reservoir that contains the cross-linking ions. The crosslinking and gelation are initiated from the outside and proceeds inwards in the alginate solution by diffusion of the ions [48, 64]. This method is suitable for creating smaller materials [48], such as alginate beads [61, 65]. To entrap cells by this method, alginate solution

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containing cells can be dripped into a calcium solution [7]. In the internal setting (Figure 6), the crosslinking ion is released homogeneously in the alginate, in a controlled manner [48]. This can be performed by mixing a calcium salt, such as CaCO3 [51], of low solubility in the alginate solution. The calcium ions can be released by adding an acid to the mix. For this, the slowly hydrolyzing acid glucono delta lactone (GDL) can be used as a proton (H⁺) donor [51, 64].

Figure 6: Internal gelation of alginate by cross-linking with Ca²⁺, in the presence of CaCO3 and GDL.

Addition of GDL causes the release of calcium ions from bound form in CaCO3. The calcium ions attach to and crosslink the alginate chains in the solution as shown in detail to the right in the figure. Illustration inspired by figure in Nobile et al., 2008 [66]. Created in BioRender.com.

Alginates can form gels at mild conditions, which makes it possible to entrap cells and proteins without disturbing biological activities considerably [67]. They can gel at RT and are heat- stable [48]. Alginate hydrogels offer a gel porosity that allows diffusion of small water-soluble molecules [67-69]. In a study by Boontheekul et al. (2005) the pore size of 1% (w/v) alginate gels was around 5 nm [70]. The gelling ability and chemical properties of the formed gel depend on, the content of G-blocks, availability of cross-linking cations and molecular weight of the chains [7, 48, 51, 56, 61]. The viscosity of the gel depends on e.g. the length and ion affinity of the alginate chains [56] and the gel composition [48]. Alginates with high G-content, which are the ones mostly applied for gel formation [7], have a high ability to form gels by Ca²⁺

crosslinking [56]. High-G alginates are often used when a high gel strength is desired. They have been said to be low on immunogenicity, compared to high-M alginates, and are often preferred for immobilization materials. [71]. However, this is debatable, for instance as shown in an in vivo biocompatibility study by Tam et al. (2011) [72] on injected alginate beads in mice, where high M-alginate resulted in lower degree of fibrosis, compared to the high-G alginate [72, 73].

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Many different alginate hydrogel scaffolds for 3D cell culture exist. Some main types are beads, gels and foams [5]. As mentioned, alginate gel beads have been used for cell entrapment. One of the most researched medical applications of alginate is the use of alginate gels in cell therapy in diabetes treatment, often in the form of microencapsulated insulin-producing islet cells for in vivo transplantation [74-76]. In this study, discs of internally gelled (Figure 6) alginate were used with cells seeded inside. Cells can also be seeded on top of such alginate gels, e.g. as shown with myoblasts in a study by Rowley et al. (1999) [77]. Alginate foams containing alginate solution were also used here. In these foams, the alginate solution is crosslinked by precipitation of calcium ions from the foam structure (Figure 7). Alginate foams and sponges are macroporous scaffolds. Their porosity may facilitate cell-cell connections and led to better diffusion of nutrients, oxygen and waste compared to an ecapsulation material without this support structure [7, 78, 79]. High porosity foams that are able to support cell infiltration can be produced by steps of internal gelation, gas foaming [80] and freeze drying [7, 80, 81].

Figure 7: Gelation of alginate foams. Calcium ions diffuse from the foam walls (lamella) (B) into the pores upon addition (A) of alginate solution and causes cross-linking (C). The cells are in solution and will be entrapped when the gelation occurs [5, 31]. Illustration copied from review article by Andersen et al. (2015) [5].

Cultivation of fibroblasts in alginate sponges has shown that the cells were immobilized inside pores and were able to proliferate. Their morphology remained spherical, as opposed to the flat morphology that is seen in monolayer cultivation [79]. Due to their increased mass transport and cell infiltration, macroporous scaffolds are especially of interest in tissue engineering [82, 83]. Porous alginate hydrogels for tissue engineering have been used for encapsulation of a hepatocarcinoma cell line (hepG2) and showed increased proliferation and larger cell spheroids under porous conditions [82].

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The foams used in this study (Figure 8) come as gamma-sterilized, dry discs prepared in well plates, and turn into hydrogels upon rehydration with alginate solution. Unfortunately, NovaMatrix^®did not provide any specifications regarding the dry alginate foams due to their termination in production. However, it is likely that the foams used for experiments in this thesis are similar to those used by Andersen et al. (2014) [31] in their study. Those were produced from 2% (w/w) alginate by ionically gelling with CaCO3 and GDL then aerated and air-dried. The foams were 2 mm thick before drying [31]. Foam pore sizes can vary from 225- 400 µm, regarding dry foam density (DFD) and alginate concentration (% w/w). Low density and low alginate concentration give larger pores. Foaming agents used were polysorbate and hydroxypropyl methylcellulose (HPMC) [29, 31]. Cells are seeded using alginate solution as a carrier, and gelation occurs when the solution with the cells comes in contact with the pore walls. Andersen et al. (2014) used these foams with 0.1%-1% alginate and obtained seeding efficiency of cell line murine fibroblasts (NIH:3T3) between 95-115%, compared to 18%

without alginate [31].

Figure 8: Dry alginate foams with interconnected pore network. Details and size of pores is shown in scanning electron microscopy (SEM) images (A and B), showing pore diameters of 352 µm, 189 µm and 48 µm. Light images show foam surface seen from above (C) and cross section (D). Scalebars in C and D are 250 µm. From Andersen et al. (2012) [29].

Foams are considered to be something between 2D and 3D, because the cells can spread on the surface of pore walls, similarly to the attachment to a surface in monolayer 2D culture [4, 5].

However, in the sponges used by Prestwich (2007) described as 2.5D, the cells are added suspended in media [4]. In the foams used in this study, the cells were mixed with alginate solution priorly. The use viscous alginate solution as carrier for cells can be highly beneficial

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in reducing the number of cells that otherwise could end up colonizing the bottom of the well plate, as seen with the increased cell seeding efficiency in Andersen et al. (2014) [31].

For cells to be able to survive and thrive in alginate materials, the materials must be modified [7]. The free hydroxyl (-OH) and carboxyl (-COOH) in the alginate allows for chemical modifications and thereby functionalization of the alginates [7, 84]. Oxidation with periodate is commonly performed on alginates. Periodate opens the ring structure and forms a dialdehyde, increasing the flexibility of the chain. This enhances degradability [7, 71]. Oxidation of alginates with periodate (IO-anion) gives more reactive groups [85] and facilitates attachment of substituents or cross-linkers [7]. The degree of oxidation allows for control of degradability [70].

For enabling cell attachment to the material, the alginate can be grafted with RGD [7]. RGD- coupling of alginate has shown to promote both adhesion and proliferation of murine osteoblast cells [86], chondrocytes [87] and myoblasts cells [77, 88]. Alginates cannot interact with mammalian cells on their own, but by covalently coupling RGD-cell adhesion ligands at free -COOH groups in the chains this is made possible. Such grafted polymers offer the possibility to explore specific cell receptor-ligand interactions [77, 89]. In addition to chemical modifications, material properties of the alginates like stiffness, degradability and topography can also affect the biological activity [7]. Degradability can be modified by grafting the alginate with MMP-cleavable peptides, allowing the cells to enzymatically degrade and remodel the gel matrix. This has shown to increase degree of cell elongation and cellular networks of MSCs [90]. Apart from degradation by cells, the stability of alginate gels is affected by presence of transition metal ions, oxygen, and pH. Exposure to acid or alkaline conditions are primary reasons for depolymerization [91].

Regarding the specifications for alginates used in this study, these were ultrapure unmodified alginate products from NovaMatrix®, with proposed applications in tissue engineering, medical devices, drug delivery and cell encapsulation [92]. The raw material for their alginates is the large brown macro algae Laminaria hyperborea [93, 94]. The unmodified alginate was used in mix with RGD-grafted alginate to make alginate solutions with cell adhesion possibility.

1.3 Cells of interest

Three different human-derived adherent cell types were used in this study, whereof two were cell lines and one was primary cells. The primary cells were normal human dermal fibroblasts (NHDFs). The cell lines were human lung fibroblasts (IMR-90s) and human bone marrow

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stromal fibroblast-like cells (HS-5s). Fibroblasts are a heterogenous family of mesenchymal- derived cells [95-97]. The fibroblasts have many morphological similarities with mesenchymal stem cells (MSCs) [98], also called mesenchymal stromal cells. MSCs are a heterogenous group of multipotent progenitor cells with a fibroblast-like spindle morphology, that can differentiate into cell types such as osteoblasts, chondrocytes, marrow stromal cells and adipocytes [46, 99- 104]. The fibroblasts are found in all tissues of the body [96], where they reside in the interstitial spaces of organs [105]. They have important functions in secreting ECM-components and interacting with the ECM [105-107], and are abundant in connective tissue [19, 97]. Fibroblasts are important players in injury responses [105]. Upon damage in tissues, fibroblasts migrate through the connective tissues and rebuilds or remodels where necessary [108]. As mentioned in Section 1.1.1, the ECM is essential in mechanical support of cells, and facilitates adhesion and migration, which all are crucial functions for cell survival and proliferation. Disorders such as fibrosis and cancer are associated with malfunctions in the cell-ECM interaction [106]. The importance of understanding functions of fibroblasts has made them very common in cell biology studies [109], and they are widely used both in form of primary cell cultures and immortalized cell lines [97].

Fibroblasts were first described as a cell type by Rudolf Virchow in 1858 [110, 111]. They are known for having spindle-like elongated morphology [97], at least in inactive form [107].

However, morphology and functions of the fibroblast varies according to tissue and state of the tissue (Figure 9) [95, 96]. This “topographic differentiation” is shown also by different gene expression profiles of fibroblasts in different anatomical locations [112]. Fibroblasts significantly change their native characteristics when they are cultivated on flat surfaces [1, 17].

Studying these cells only in 2D therefore might lead to biological properties being missed.

Grown in 2D, their ECM receptors assemble on the ventral side towards the culture surface and the cell adopts a highly spread, flat shape with large lamellipodia and high degree of stress fibers [17, 113]. (Stress fibers and formation of lamellipodia are further explained in Section 1.3.2.) On the other hand, in 3D cultivation, the receptors are spread all over the cell, and this can give a more global interaction that might allow a highly elongated and less flat-spread morphology [17, 113, 114].

Cultured fibroblasts have shown to differentiate into myofibroblasts, especially when grown in low cell densities. Myofibroblasts have smooth-muscle-like features and have an important role in wound healing by contracting the wound [95, 115-117]. There are studies and reviews that have indicated that there are many phenotypical similarities between fibroblasts and MSCs,

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including when it comes to abilities in differentiation into tissue specific cell types [98, 118, 119]. However, they seem to have some differences in gene expression and regulation. The exact relation between the two groups of cells is a complex topic of debate, indicating that there still is a need for further characterization of these cells [98]. This will therefore not be addressed further in this thesis, where fibroblastic cells are in focus.

Figure 9: Early drawings of fibroblasts by Rudolf Virchow, Ramón y Cahal Santiago and Ernst Ziegler, showing A) spindle-shaped cells in pig embryo connective tissue, B) fusiform cells in keloid scarring, and C) various forms of fibroblasts in new connective tissue upon tissue healing [110, 111, 120, 121]..

Obtained and modified from review by Plikus et al. (2021) [110].

The home of the fibroblasts, the connective tissue, is one of the four types of animal tissue. It consists of cells scattered through an ECM that varies from liquid to gel-like to almost solid, and is found across adipose, cartilage, bone, and blood bearing organs [122]. The main roles of connective tissue includes mechanical support, holding tissues and organs in place [107, 122].

It also functions as nutrients storage, holds blood vessels and allows movement of cells and nutrients [107]. Resident cell types are fibroblasts, adipocytes and mast cells, but it is also often home to free blood cells. Different types of connective tissue vary in composition, type and content of ECM-components and cells [107]. Connective tissue comprises loose connective tissue and dense connective tissue [107, 122]. Loose connective tissue is found in lamina propria of digestive- and respiratory tracts and epithelial linings of organs, where it permits diffusion of fluids and movement of cells. Dense connective tissue provides mechanical support and are collagen rich. It can be either irregular or regular, based on whether the thick collagen bundles are randomly arranged or arranged unidirectionally. The irregular is found in dermis of skin, and encapsulates organs, and its fibroblasts are usually inactive and compressed. The regular is found mostly in tendon and has fibroblasts that usually exhibit spindle-shape [107].

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All three cell types used in this study are adherent human fibroblasts or fibroblast-like cells derived from dermis of skin, lung tissue or bone marrow. The fibroblasts from dermis, NHDFs (Figure 10), were primary cells from adult human skin. According to supplier (Lonza) they are suitable for applications such as ECM protein analysis, wound healing, collagen metabolism, cosmetics, skin therapy and skin models. They are guaranteed 15 population doublings [123].

Figure 10: Adult NHDFs in culture, stained with immunohistochemistry in 24-wellplate (left) and in culture at high density showing a uniform morphology (right). Images by suppliers (Lonza Products) [123].

Dermis is the middle layer of skin, between the upper layer of epidermis and bottom layer of subcutaneous fat [124]. It makes up the largest part of the skin and provides mechanical support [124, 125]. The dermal fibroblasts (Figure 10, Figure 11) are either papillary, reticular or follicular, and have functions in remodeling and repairing wounds and maintaining the skin physiology [125]. Dermal fibroblasts are widely used in research, and is often considered the most suitable cell type for studying cell metabolism under normal conditions, during development and aging, upon disease or exposure to agents [126].

Figure 11: Dense irregular connective tissue of human dermis showing a fibroblast (dark area) surrounded by ECM high in collagen fibers (light area). Magnification is x10 000. Image from Ultrastructure Atlas of Human Tissues (2014) [107].

Figure 12 shows IMR-90 in 2D culture. IMR-90 is a fibroblast-like cell line originating from human lung tissue. It was derived by W.W Nichols et al. in 1975 from the lungs of a 16-week-

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old aborted female fetus, for use in research [127-129]. The cell line is banked in large quantities in support of the National Institute on Aging (NIA) research and details were obtained from American Type Culture Collection (ATCC). According to ATCC the cell line is for laboratory research only, and suitable for 3D cell culture and in use as a transfection host. The IMR-90 cell line is reported to be capable of 58 population doublings [127-129].

Figure 12: Light microscopy images of IMR-90s in culture with low density (left) and high density (right). Scalebars are 100 µm. Images from ATCC [127].

Lung tissue, belonging to the respiratory organ system [122], consists of the upper and lower system. The nose, paranasal sinuses and pharynx belongs to the upper, while the lower system comprises trachea, bronchi and bronchioles [130]. Connective tissue gives stability and helps in the functions of the lung [131]. It is found in the interstitial spaces, parenchyma, and the most common cells in this interstitium are fibroblasts [132]. The lung fibroblasts are usually located close to the lung epithelium or endothelium [105], and have functions in the elasticity that is needed for breathing [110]. They are important for supporting the alveolar structure, both by proliferating and by repairing damage [133]. Diseases in the connective tissue can give many different complications [134]. In lung diseases such as asthma, fibrosis and chronic obstructive pulmonary disease (COPD), there are changes in the fibroblasts in terms of numbers and characteristics [105]. As mentioned, IMR-90 is from a 16-week-old embryo [127-129]. The developmental stage of lungs in a 7–17-week-old embryo is called the pseudoglandular stage, and involves differentiation of epithelial cells, and development of airways, bronchioles, pulmonary arteries and veins. The lung is still immature at this point. While still in development, airways are lined by undifferentiated cells and the interstitial space is large and has low vascularization [130].

Different from the other cell types, the HS-5 cell line (Figure 13) has been transformed with a virus for immortalization. The cell line origin is stroma of bone marrow from a 30-year-old

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male, and it was isolated by Fred Hutchinson Cancer Research Center in 1995. According to ATCC, usages for this cell line are in laboratory research purposes only [135]. The cell line is suitable for 3D cell culture and immunology studies, as feeder layer in ex vivo bone marrow cultures or in colony forming assays. The virus used for immortalization was human papilloma virus (HPV)-16 E6/E7, and the cell line was transformed with the HPV-sequences using a retrovirus vector. HS-5 is the fifth of 27 clones that were isolated. These details on the cell line was obtained as recorded by ATCC [135]. The immortalization was performed using a recombinant retrovirus containing the E6/E7 genes of the HPV. These genes interfere with tumor suppressor proteins, which causes prevention of cell cycle arrest. Of the 27 immortalized clones, HS-5 and HS-21 are fibroblast-like. HS-5 appears relatively small and forms a network of overlapping cells, that are similar to astrocytes. In high densities, HS-5 forms a dense net [136].

Figure 13: Light microscopy images of HS-5 in culture with low density (left) and high density (right).

Scalebars are 100 µm. Images from ATCC [135].

Bone marrow is a large organ located in the cavities of bones, where it is supported by porous bone microarcitectures [137, 138]. It belongs to the immune and lymphatic organ system [122]

and houses may different cell types in a viscous microenvironment. The soft bone marrow tissue [138] provides structural and physiological support for hematopoietic cells [139]. Bone marrow is the main source of the hematopoietic stem cells (HSCs), which differentiates into different blood- and immune cells, giving a continual renewal of these cells in blood [137, 138]. Adult bone marrow also contains MSCs, which are able to differentiate into bone, cartilage and adipocytes [138].

HS-5 is a fibroblast-like cell that secretes different stimulating factors and interleukins (ILs), which makes it able to support proliferation of HSCs when these are co-cultured [104, 136]. An evaluation of HS-5, HS-27 and primary bone marrow MSCs by Adamo et al. (2020), it was

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found that bone-marrow derived commercial cell lines such as HS-5 and HS-27 are good candidates as disease models for MSCs. The findings suggested HS-5 to have an expression pattern similar to bone marrow-derived MSCs, and therefore might function as a model for reproducing properties of these. As cell lines are easier to manipulate in vitro compared to primary MSCs, these findings have many advantages. The same study showed that the HS-5 cells had higher ability to differentiate into osteoblasts than adipocytes [104]. Similarly, Kabrah et al. (2015) also showed that HS-5 is suitable as an alternative for MSCs in a 3D model [140].

1.3.1 Primary cells and cell lines

An important difference between NHDF, IMR-90 and HS-5, is that the former are primary cells while the two latter are cell lines. Primary cells are cells that have been isolated directly from the tissue of interest, not yet sub-cultured. Therefore, they usually still exhibit many of their natively differentiated properties when cultured in vitro [141-144]. For instance, fibroblasts will continue secretion of collagen, epithelial cells will form epithelial-like sheets, embryonic skeletal muscle form muscle fibers, and nerve cells will form axons. Studying these cells in vivo can be difficult, so this is very favorable [141]. Fibroblast-like primary cells usually grow well and fast in culture [142]. Primary cells can be passaged for weeks or months. However, their capacity for division is finite and they will eventually die. Normal human somatic fibroblasts can usually divide 25-40 times before they stop [141] due to replicative cell senescence [141, 142, 144, 145]. Replicative cell senescence is caused by continual shortening and uncapping of the ends of the chromosomes (telomeres) and leads to the cells having a limited replicative life span. The telomeres are repeated DNA-sequences with protein caps on the end of chromosomes. This shortening leads to a decline in cell division rate and other changes in cell characteristics [141, 145-147], and is therefore an important limitation with the use of primary cells [142]. In addition, primary cells are sensitive and have specific requirements when it comes to cultivation media and supply of growth factors and nutrients, which can be challenging. Some primary cells will differentiate along their native lineage after each passaging (self-replication), while others, like primary hepatocytes, instead tend to dedifferentiate and lose their characteristics [144].

Replicative senescence seems to be a tumor suppressor mechanism, in the way that tumors often have mutations that allow them to overcome this. Many malignant tumor cells can replicate continuously, as well as some stem cells [141, 145-147]. The obstacle of replicative senescence can be overcome by creating immortalized cell lines by transformation. Telomerase is an enzyme that maintains the telomers, but which is not expressed in human somatic cells. By

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transforming human cells with the gene that encodes telomerase, immortalization can be obtained. Such transformed cell lines can grow in higher densities in culture than normal primary cells, and they can sometimes also grow without needing attachment. Cell lines can be generated from cancer cells, or by transforming normal cells [141]. There are different ways to transform cells for immortalization, for example transfection with tumor-inducing chemicals, or with viruses, such as the viral HPV E6/E7 genes, or by transfection with human telomerase [141, 144]. There are many different cell lines that exist, and one of the most famous is the first one created, the HeLa cells, originated from human epithelial [141].

Immortalized cell lines are available and can be expanded almost without limitation [148].

However, they are likely to differ more from in vivo state than primary cells do [141, 148]. Pan et al. (2009) compared mouse primary hepatocytes to a mouse hepatoma cell line (Hepa1-6).

Even though many important signaling pathways were maintained in the cell lines, many proteins were down-regulated and there were differences in characteristic metabolic pathways, the immune system and in ECM-synthesis. The cell line showed a shifting focus to proliferative functions. However, the study only involved primary cells at a specific time point and a specific cell line [148]. Though cell lines don’t guarantee an accurate resemblance of primary cells, they have been very important in, among other things, vaccine research, drug development and testing, gene studies and tissue engineering, due to their availability and ease of use. They also allow avoidance of the ethical concerns with obtaining cells from human and animal tissue [149].

1.3.2 Cytoskeletal response to the microenvironment

Different tissues have different properties regarding stiffness and elasticity (Figure 14). Skin, muscle and brain are examples of soft tissues that have elasticity due to the presence of the adherent cells in the ECM. Tissue cells can sense the stiffness of their environment, and evidence suggests that this information affects the properties of adhesion and cytoskeleton behavior [24]. It seems that the stiffer the surroundings are, the higher the stiffness of the cell is [150].

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Figure 14: Stiffness in terms of elastic modulus of tissues in the human body, measured in pascals (Pa).

Brain and lung are amongst the softest tissues, while stromal tissue (fibroblasts) is closer to the middle.

Most tissues are not nearly as stiff as stiff as plastic and glass, which are commonly used substrates for 2D cell culturing. Figure from Cox and Erler (2011) [150, 151].

The interaction between cells and their surrounding matrix depends on signaling that is ligation- induced, such as binding of integrin to fibronectin-RGD and traction-induced, based on mechanical stimuli [38]. However, these go together, since the mechanical signaling is transduced via the binding of integrin [109]. Through mechanotransduction, integrins translate mechanical cues from the extracellular environment to internal signaling. Human MSCs have shown to sense their mechanical environment this way.When cultured on substrates with tissue- specific matrix properties, MSCs differentiated into specific cell types. With stiffnesses around 100 Pa, 10 000 Pa and 25 000 Pa, they showed differentiation towards neurons, skeletal muscle cells and bone cells, respectively [45]. This indicates that modified matrices can be used to differentiate MSCs. For bone cells, microporous scaffolds might be better, while for soft tissue cells, hydrogels might be more suitable [3].

The way cells can sense the stiffness of a material they have attached to, is through pulling forces. The pulling triggers an internal response in the cell and causes recruitment of integrins and other proteins to the binding site. This response is strong when the attached matrix is rigid, while attachment to a soft matrix will give less tension in the cell and a lower response [19].

For a cell to be able to move, it must have the possibility to exert such a traction on the ECM, adjacent cells or a culturing substrate [39]. The pulling forces are imposed by actin-myosin contractions in the cytoskeleton [24], and allow the cell to move along extracellular fibers [109].

Adherent cells apply force to their surroundings, and respond to their surroundings through cytoskeletal organization [24]. Changes in the cytoskeleton triggers pathways in the cell and causes changes in the cell [109] according to the microenvironment [42].

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The cytoskeleton is a fibrous protein network that extends throughout the cytoplasm of the cell.

It provides mechanical support, anchorage, and transport for cell organelles, and consists of actin filament, intermediate filaments, and microtubules. Actin filaments, found all over the cell, consist of two intertwined strands of globular actin units [109]. It is the actin filament that exert pulling forces [109]. Actin filaments occur as single filaments, in linear bundles, 2D networks and 3D gel-like structures (Figure 15). Cell movement and morphology are regulated by the formation of actin filament, which occurs through a nucleation driven process [108].

This involves a complex reorganization of the cytoskeleton [42, 108]. The cells spread or move by protrusion of the cell membrane by actin filament nucleation, dynamic attachment to the extracellular material and actin-myosin contraction. In the latter step, the cell can be drawn forward. The cell protrusions can have different shapes, but common in fibroblasts are filopodia and lamellipodia. Filopodia are spike-like, one-dimensional projections and contain a core of long, bundled actin filaments. These allow the cells to explore the environment. Lamellipodia are two-dimensional, sheet-like structures with crosslinked actin filaments [108].

Figure 15: The different actin filament (red) arrangements in a cell, here illustrated by a fibroblast crawling on a 2D tissue-culture dish. The arrows on the actin filaments points towards the minus end of the filament, which is the slow-growing end [108].

The stress fibers are contractile actin filament bundles that terminate in the focal adhesion, connects with the ECM and exert tension [108, 152]. The focal adhesion is the area of attachment where the actin filament is linked with the ECM. In a dynamic manner, these adhesions are formed at the leading edge and disassemble at the back as the cell moves forward [108, 153]. Focal adhesions appear as integrin associates with ECM-ligands, which leads to further clustering of integrins and other proteins. This allows the transmission of information between ECM and cell cytoplasm [6, 152]. Through the tension caused by actin-myosin

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activity, the strong interaction in the focal adhesions exerts a pull on the substratum the cells are attached to and allows movement of the cell [108, 152]. Focal adhesions eventually become elongated structures, aligned with the stress fibers [153].

The extent to which cells generate stress fibers and focal adhesions is largely dependent on the properties of the substrate itself [17, 18, 24, 38, 42, 113, 150]. For instance, SMCs have shown to have less stress fibers and focal adhesions in vitro in a 3D matrix than in 2D culture [18].

Because of this, in addition to receptor-ligand interactions, cell functions will also depend on mechanical properties of their environment, such as stiffness, but also topography and architecture [154]. Properties on the nanoscale (less than 1 μm), microscale (1-1000 μm), like porosity and pore size, and macroscale (bigger than 1 mm), like scaffold dimension, can be of importance [155]. The size, geometry and topology of pores are physical cues that can guide cell morphogenesis, and which also are important for the spatial organization cells. Grooves and roughness in surfaces have shown to affect cell orientation and can also improve attachment of some cells. Fibroblasts have shown to prefer smooth surfaces, while osteoblast-like cells prefer rougher surfaces [156]. In a bone marrow cell line (SR-4987) the spatial development was investigated by culture in three different porous materials, two different porous cellulose beads (100 μm and 500 μm pore size) and a polyester fibrous material. This resulted in growth that was globular, spread, and thin and long, respectively [156, 157].

1.4 Methods for studying cells cultivated in 3D scaffolds

In this study, viability and morphology was studied using light microscopy, confocal laser scanning microscopy (CLSM) and a cytotoxicity test based on release of the cytosolic enzyme lactate dehydrogenase (LDH). CLSM was chosen as the main method of analysis due to its suitability for studying cells in three dimensions. CLSM requires fluorescence staining prior to imaging and was used here for differentiating live from dead cells, and for staining specific parts of the cell. Light microscopy was used for studying the cells while they were still alive, and the cytotoxicity test complements the viability imaging.

Light microscopy requires no fixing or staining, and therefore allows studying cells while they are alive. In bright-field light microscopy, the image is formed directly by light passing through the specimen [158]. To improve contrast when examining unpigmented cells, phase-contrast can be applied. One can also stain the cells, but this would involve killing them [158]. The light microscope can separate details that are 0.2 µm apart. If they are closer, they will appear as a single object. When using the light microscope, the focus is on a particular plane of choice in

Viability and morphology of human fibroblasts in RGD-functionalized 3D alginate scaffolds Qualitative image analysis with focus on the effects of microstructuring

Andrea Mandt-Utbøen Holtermann

Viability and Morphology of Human Fibroblasts in RGD-functionalized 3D- Alginate Scaffolds

Qualitative Image Analysis with Focus on the Effects of Microstructuring

Master ’s thesis

Andrea Mandt-Utbøen Holtermann

Viability and Morphology of Human Fibroblasts in RGD-functionalized 3D- Alginate Scaffolds

Qualitative Image Analysis with Focus on the Effects of Microstructuring

Acknowledgements

Abstract

Sammendrag

Abbreviations

Contents

1 INTRODUCTION